AmineResults Luciferase Reporter Assay SystemIn order to find ligands for hTAAR

AmineResults Luciferase Reporter Assay SystemIn order to find ligands for hTAAR5 we employed a 23388095 Creluciferase reporter gene assay in which TAAR receptor activation leads to an elevation of cAMP and the subsequent expression of luciferase as described for odorant receptors [13]. We transfected HANA3A cells with cDNA coding for a rho-tagged hTAAR5 together with Golf and RTP1S to ensure functional expression of the receptor, and the cAMP dependent reporter gene construct Cre-luciferase [14]. Presence of the expressed receptor protein was tested by immunocytochemical detection of the extracellular Nterminal rho-epitope tag in fixed HANA3A cells (Fig. 1) and by live-cell staining on the surface of HANA3A cells (GDC-0853 Figure S1). In total, 42 amines or amine related substances were tested for an agonistic action on hTAAR5 (Materials and methods). Transfected cells were stimulated by 100 mM of the test substances for 4 h and induced luciferase activity was assayed by luminescence activity. Tested repertoire (Materials and methods) first based on the volatile amines known as agonist for the murine “olfactory TAARs” [2] and trace amine ligands for h/mTAAR1 [1,15]. After finding out that hTAAR5 can be activated by TMA, we further tested chemical analogs of TMA. The chemical variations range from primary amines (methylamine) to quaternary amines (tetramethylammonium hydroxide) or diamines (putrescine) as well as to the substitution of nitrogen by phosphate or the substitutions of methyl groups by longer aliphatic chains, which form acyclic-, cyclic-, heterocyclic- or aromatic amines (Fig. 2). All chemical variants tested, abolished the activity on the hTAAR5 receptor as shown by the lack of signal increase (Fig. 3). Furthermore, the natural occurring trimethylamine N-oxide was inactive in concentrations up to 1 mM (data not shown). Only the tertiary amines TMA (100 mM) and the substitution from one methyl group by one ethyl group, namely dimethylethylamine(DMEA) (100 mM), significantly (p,0.001) activated hTAAR5 expressing cells (Fig. 3). In subsequent experiments we constructed concentrationresponse curves of the two active substances that revealed that 1 mM TMA is sufficient to induce significant signals above detection threshold (p,0.05). Adding of 1 mM TMA to the extracellular media led to the induction of a strong luciferase activity that was even higher than the signal induced by the adenylate cyclase activator forskolin (10 mM) as positive control. TMA is the most potent hTAAR5 ligand with an EC50 value of 116 mM (n = 2?3), followed by DMEA EC50 = 169 mM, n = 2?) (Fig. 4). DMEA activates hTAAR5 with a lower efficacy and is therefore a partial agonist. To compare the receptor affinities we additionally expressed mTAAR5 in HANA3A cells and measured receptor activity in the Cre-luciferase assay (Figure S2). The murine TAAR5 is more sensitive than the human 24786787 ortholog. Calculated EC50 value is 940 nM (n = 2?).Human TAAR5 Expression in Xenopus laevis OocytesDue to the fact that co-expression of different proteins like RTP1S (Materials and methods) can alter the surface receptor expression and sensitivity of the used reporter system, EC50 values measured by only one expression system have GDC-0152 limited reliabilities for statements about general receptor sensitivity. We used a different recombinant expression system to validate our data regarding the hTAAR5 sensitivity for the activating tertiary amines TMA and DMEA obtained by CRE-luciferase assay. We heterol.AmineResults Luciferase Reporter Assay SystemIn order to find ligands for hTAAR5 we employed a 23388095 Creluciferase reporter gene assay in which TAAR receptor activation leads to an elevation of cAMP and the subsequent expression of luciferase as described for odorant receptors [13]. We transfected HANA3A cells with cDNA coding for a rho-tagged hTAAR5 together with Golf and RTP1S to ensure functional expression of the receptor, and the cAMP dependent reporter gene construct Cre-luciferase [14]. Presence of the expressed receptor protein was tested by immunocytochemical detection of the extracellular Nterminal rho-epitope tag in fixed HANA3A cells (Fig. 1) and by live-cell staining on the surface of HANA3A cells (Figure S1). In total, 42 amines or amine related substances were tested for an agonistic action on hTAAR5 (Materials and methods). Transfected cells were stimulated by 100 mM of the test substances for 4 h and induced luciferase activity was assayed by luminescence activity. Tested repertoire (Materials and methods) first based on the volatile amines known as agonist for the murine “olfactory TAARs” [2] and trace amine ligands for h/mTAAR1 [1,15]. After finding out that hTAAR5 can be activated by TMA, we further tested chemical analogs of TMA. The chemical variations range from primary amines (methylamine) to quaternary amines (tetramethylammonium hydroxide) or diamines (putrescine) as well as to the substitution of nitrogen by phosphate or the substitutions of methyl groups by longer aliphatic chains, which form acyclic-, cyclic-, heterocyclic- or aromatic amines (Fig. 2). All chemical variants tested, abolished the activity on the hTAAR5 receptor as shown by the lack of signal increase (Fig. 3). Furthermore, the natural occurring trimethylamine N-oxide was inactive in concentrations up to 1 mM (data not shown). Only the tertiary amines TMA (100 mM) and the substitution from one methyl group by one ethyl group, namely dimethylethylamine(DMEA) (100 mM), significantly (p,0.001) activated hTAAR5 expressing cells (Fig. 3). In subsequent experiments we constructed concentrationresponse curves of the two active substances that revealed that 1 mM TMA is sufficient to induce significant signals above detection threshold (p,0.05). Adding of 1 mM TMA to the extracellular media led to the induction of a strong luciferase activity that was even higher than the signal induced by the adenylate cyclase activator forskolin (10 mM) as positive control. TMA is the most potent hTAAR5 ligand with an EC50 value of 116 mM (n = 2?3), followed by DMEA EC50 = 169 mM, n = 2?) (Fig. 4). DMEA activates hTAAR5 with a lower efficacy and is therefore a partial agonist. To compare the receptor affinities we additionally expressed mTAAR5 in HANA3A cells and measured receptor activity in the Cre-luciferase assay (Figure S2). The murine TAAR5 is more sensitive than the human 24786787 ortholog. Calculated EC50 value is 940 nM (n = 2?).Human TAAR5 Expression in Xenopus laevis OocytesDue to the fact that co-expression of different proteins like RTP1S (Materials and methods) can alter the surface receptor expression and sensitivity of the used reporter system, EC50 values measured by only one expression system have limited reliabilities for statements about general receptor sensitivity. We used a different recombinant expression system to validate our data regarding the hTAAR5 sensitivity for the activating tertiary amines TMA and DMEA obtained by CRE-luciferase assay. We heterol.